Replacement of the Draize rabbit eye test for ocular irritancy testing remains an elusive goal. Although monolayer cultures and stratified corneal epithelial tissues have been developed and are useful as initial screens for ocular irritancy, they are not capable of completely modeling the interaction of exogenous chemical with human corneal tissue. For instance, protocols developed with current tissue models correctly identify a high percentage of ocular irritants (~ 95%) but misclassify a large percentage of non-irritants as irritants (~33%). Also, these models are not capable of modeling extent of injury and time to recovery following chemical insult. Thus, animal or human clinical tests are still needed to gain a compete picture of the effects of chemical exposure to the eye. The current application will develop a corneal full thickness (CFT) tissue model compromised of endothelial, stromal, and epithelial components. The CFT tissue will contain human cells and develop tissue barrier properties that are comparable to native corneal tissue. Because of its improved barrier, the CFT will avoid the misclassification of non-irritants. In addition, the CFT tissue will be useful in determining the extent of injury and time to recovery following noxious chemical exposure. Due to these enhanced capabilities, the CFT will provide toxicologists and scientists with a tool that will dramatically decrease, if not eliminate entirely, the need for animal testing to determine ocular irritancy. PUBLIC HEALTH RELEVANCE: Safety assessment of all new chemicals and products requires evaluation of potential ocular irritancy in case of intentional or unintentional exposure to the eye. Current animal-based test methods are sup-optimal because of animal welfare concerns and in vitro methods lack the complexity required to completely model in vivo responses. We will develop a corneal full thickness tissue and in vitro test methods that will dramatically reduce or eliminate the need to use animals to assess ocular irritancy of chemicals and products. [unreadable] [unreadable] [unreadable]